Activation of the RAF/mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway mediates apoptosis induced by chelerythrine in osteosarcoma.
OBJECTIVE
Chelerythrine, a widely used broad-range protein kinase C inhibitor, induces apoptosis in many cell types. In this study, the mechanism of chelerythrine-induced apoptosis in osteosarcoma was investigated.


METHODS
Signaling pathways activated by chelerythrine in osteosarcoma were detected by Western blots. Impacts of RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK on apoptosis and cell survival were studied using genetic approaches and pharmacologic pathway-specific inhibitors.


RESULTS
Osteosarcoma cells underwent apoptosis rapidly after treatment with chelerythrine. Three parallel MAPKs pathways, including the ERKs, c-Jun NH(2) kinases, and p38, were activated by chelerythrine in a dose-dependent and time-dependent fashion. For the ERKs, the activation was evident at the earliest time point tested (2 minutes) and sustained for >4 hours. Introduction of a dominant-negative H-RAS mutant (17N) partially attenuated ERK activation and delayed the onset of apoptosis induced by chelerythrine. The ERK activation and apoptotic effects of chelerythrine were greatly abrogated by the pharmaceutical inhibitors of MEK, but not by those of c-Jun NH(2) kinase or p38. Moreover, osteosarcoma cells were sensitized to chelerythrine by transient transfection with wild-type MEK1 or constitutively active MEK1 and became resistant with dominant-negative MEK1. Other protein kinase C inhibitors, including GF109203X or Gö6976, did not cause ERK activation or apoptosis in the same timeframe tested.


CONCLUSIONS
In osteosarcoma, chelerythrine-induced apoptosis is mediated through activation of the RAF/MEK/ERK pathway. These findings suggest that activating the ERK MAPK, as opposed to inhibiting it, may be a therapeutic strategy in osteosarcoma.
Yang
Rui
R
Department of Pediatrics and Molecular Pharmacology, The Albert Einstein College of Medicine, The Children's Hospital at Montefiore, Bronx, New York 10467, USA.
Piperdi
Sajida
S
Gorlick
Richard
R
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D053119:Benzophenanthridines / Q000494:pharmacology*; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011905:Genes, ras / Q000502:physiology; D006801:Humans; D020287:In Situ Nick-End Labeling; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D053119:Benzophenanthridines; C016299:chelerythrine; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases